J>  • 


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r.  S.  DEPARTMENT  OF  AGRICULTURE, 
BUREAU  OF  BHTOMOLOGY— CIRCULAR  No.  157. 

L.  O.  HOWARD.   1  nlomologwl  .ndChirf  of  Bur«u. 


THE  CAUSE  OF  EUROPEAN  FOUL  BROOD. 


G.  1-    WHITE,  M.  I)..  Ph.  D. 


i 


VlvERKliENT  PRINTING  OTFICE  :  t»II 


BUREAU  OF  ENTOMOLOGY. 

L.  o.  Howakd,  Entomologist  and  Chief  <>f  Hunan. 

('.  L.  Mablatt,  Entomologist  and  Acting  Chief  in  Absence  of  chief. 

li.  s.  Clifton,  Executive  Assistant. 

W.  V.  Tastet,  Chief  clerk. 

F.  II.  Chittenden,  in  charge  of  truck  crop  and  stored  product  insect  in  vt  litigations. 

A.  D.  Hopkins,  in  charge  of  forest  insect  investigations. 

\V.  I>.  Hunter,  ;'//  charge  of  southern  field  crog  insect  investigations. 

F.  M.  Webster,  in  charge  of  cereal  anil  forage  insect  investigations. 

A.  L.  Quaintance,  in  charge  of  deciduous  fruit  insect  investigations. 

E.  F.  Phillips,  in  charge  of  bee  culture. 

D.  M.  Rookks,  in  charge  of  preventing  spread  of  moths,  field  work. 

Uoi.i.a  V.  CURRIE,  in  charge  of  editorial  work. 

Mabel  Colcord,  in  charge  of  library. 

Investigations  in  Bee  Culture. 

B.  F.  Phillips,  in  eliargc. 

G.  F.  White,  J.  A.  Nelson,  experts. 

G.  S.  Demutii.  A.  II.  McCbay,  X.  E.  Mcindoo,  apicultural  assistants. 

Pearle  II.  Garrison,  prcparator. 

H.  A.  Surface,  I).  B.  Casteel,  collaborators. 


Circular  No.  157.  toned  M  i 

United  States  Department  of  Agriculture, 

BUREAU   OF   ENTOMOLOGY. 
L.    O.    HOWARD.    Entomologist  and    Chief  of   Bureau. 


TIIK  CAUSE  OF  EUROPEAN  FOUL  ItKOOO. 

Bj  G    l'   White,  M.  l>..  Ph.  I».. 
pert  in  Bacteriology. 

HISTORIC  \i.. 

The  purpose  of  this  preliminary  paper  is  to  discuss  briefly  the  ex- 
citing cause  of  European  foul  brood. 

With  diseased  materia]  furnished  by  Cheshire,  Cheyne  made  a 
bacteriologica]  study  of  foul  brood.  The  latter  isolated  and  described 
;i  bacterium  from  the  brood  dead  of  the  disease  and  identified  it  as 
/,'  /7///.v  alvei.  Cheshire  agreed  that  the  identification  was  correct. 
A  joint  paper  by  these  men  appeared  in  lNS-">.  and  for  about  a  decade 
and  a  half  thereafter  Bacittw  alvei  was  generally  considered  to  lx> 
the  cause  of  foul  brood.  The  disease  which,  it  i-  believed,  Cheyne 
Btudied  is  the  one  that  has  received  the  designation  European  foul 
brood. 

This  disease  is  also  believed  to  lie  the  one  which  William  R.  Howard 
worked  upon  and  named  "  black  brood."  In  1900  be  described  as 
it-  cause  a  bacterium  to  which  he  gave  the  name  BadUtU  miffi. 

It  i>  probable  that  Burn  in  1906  was  studying  the  same  disease  in 
Sw  itzerland  when  he  referred  to  "  sour  brood."  Iii  the  disease  he  dis- 
covered a  bacterium  to  which  he  referred  a-  the  u  gunthi  ri-fonns." 

Maa—eii  in  L907,  working  probably  with  the  same  disease,  appar- 
ently encountered  the  u gwUheri— forms "  reported  by  Burn  and 
named  the  new  species  Streptocoa  us  n, 

-mMi    i  mm  hi:  WORK  uv  THE  WRITER  ON  El  BOFKAH   i  "l  i.  BROOD. 

In  1907  the  writer  observed  in  European  foul  brood  the  bacterium 
which  bail  been  named  Strepi  apt*.     It  was  observed  at  the 

same  time  that  there  was  present  also  another  microorganism  quite 

1 


2  THE   CAUSE   OF   EUROPEAN    FOUL  BROOD. 

similar  in  appearance,  but  clearly  different  from  the  one  which  Burri 
had  observed  and  Maassen  had  named.  All  attempts,  however,  to 
cultivate  this  new  species  were  unsuccessful.  Until  more  was  known 
about  this  organism  it  was  referred  to  in  1908  as  "Bacillus  F." 

In  1!)07  the  writer  demonstrated  that  American  foul  brood,  an 
infectious  brood  disease,  could  be  produced  by  feeding  to  healthy 
colonies  pure  cultures  of  Bacillus  larva.  This  fact  emphasized  the 
probability  that  if  European  foul  brood  is  also  caused  by  a  bac- 
terium this  disease,  too.  could  be  produced  by  feeding  pure  cultures 
of  the  bacterium  causing  it. 

To  demonstrate  this,  it  was  desirable  in  the  first  place  to  determine 
whether  or  not  the  virus  of  European  foul  brood  was  present  and 
active  in  the  diseased  brood.  Healthy  bees  were  fed  sirup  which 
contained  a  suspension  of  the  diseased  brood,  and  European  foul 
brood  was  produced.  This  showed  that  the  diseased  brood  did  con- 
tain the  virus  and  that  the  disease  could  be  produced  by  feeding. 
This  being  done,  pure  cultures  of  Bacillus  alvei  isolated  from  the  dis- 
eased material  were  substituted  for  the  diseased  brood  in  the  inocu- 
lation experiment.  Pure  cultures  of  Streptococcus  apis  were  isolated 
and  used  likewise.  Then  cultures  of  Bacillus  alvei  and  cultures  of 
Streptococcus  apis  were  used  simultaneously  in  making  the  inocula- 
tions. European  foul  brood  was  not  produced  in  any  of  the  experi- 
ments where  pure  cultures  of  either  or  both  organisms  were  used. 
These  facts  were  sufficient  to  eliminate  tentatively  Bacillus  alvei  and 
Streptococcus  apis  from  the  list  of  possible  causes  of  European  foul 
brood,  and  to  justify  a  strong  suspicion  that  the  microorganism  which 
was  referred  to  as  "  Bacillus  Y  "  bore  a  causal  relation  to  the  disease. 
It  was  necessary,  however,  to  reckon  with  other  factors  before  a 
more  definite  statement  could  be  made. 

A  continuation  of  the  work  on  the  cause  of  European  foul  brood 
has  yielded  some  interesting  results.  These  will  be  briefly  con- 
sidered in  this  preliminary  paper. 

It  has  been  observed  in  the  examination  of  diseased  brood  that 
Bacillus  alvei  is  frequently  either  absent,  or  present  only  in  small 
numbers,  in  many  of  the  larvae  which  seem  from  gross  appearance  to 
be  dead  of  European  foul  brood.  Such  samples  have  been  received 
as  a  rule  from  localities  in  which  apparently  the  disease  had  only 
recently  appeared.  Frequently,  also.  Streptococcus  apis  seemed  to 
be  absent,  or  present  only  in  small  numbers,  in  many  of  the  larva? 
which  from  gross  appearance  gave  strong  evidence  of  European  foul 
brood.  These  bacteriological  findings  further  strengthened  the 
theory  that  neither  Bacillus  alvei  nor  Streptococcus  apis  is  the  pri- 
mary exciting  cause  of  European  foul  brood.  Other  inoculation 
experiments  were  performed,  using  pure  cultures  of  these  two  species. 


Till     CAU8I     "i     i.i  K'Ti  \n    lori.   BB ». 

The  results  were  always  negative,  confirming  further  similar  results 
that  had  been  previously  obtained. 

I.i  '  i  \  r  WORK    l'.\     I  Ml     WHITER. 

While  these  facts  were  in  m  measure  satisfactory,  as  i « - 1 i « ' i i •  ^r 
strongly  in  indicate  certain  conclusions,  there  was  -till  wanting 
that  degree  of  conclusiveness  which  i-  always  desired.  By  exp< 
mental  inoculation  and  by  the  studj  of  the  brood  sick  or  dead  of  the 
disease  which  was  artificially  produced,  however,  considerable  in- 
formation  of  the  character  hoped  for  has  been  obtained.  The 
detail-  of  the  technique  used  in  making  the  inoculation  will  nol  be 
given  in  this  brief  report. 

I  KFEBIM1  Mil.   I  mm  I  i  \  i  iiiw. 

Diseased  material  from  various  localities  was  used  for  these  inocu- 
lations. Some  colonic-  were  fed  diseased  brood  that  contained,  as 
revealed  by  cultural  examinations,  large*  quantities  of  Bacillus  nl >■<'*; 
oilier  colonic-  were  fed  diseased  material  containing  large  numbers 
of  Streptococcus  apis  as  shown  by  culture-,  and  -till  others  were 
fed  diseased  materia]  which  was  demonstrated  to  contain  neither 
/;  illus  ill '■<  i  nor  Streptococcus  apis.  It  was  found  that  at  the  first 
appearance  of  the  disease  in  each  class  of  experiments  the  symptoms 
manifested  by  the  3ick  larva?  wen-  the  same.  Larvae  showing  thi 
early  symptoms  were  studied  bacteriologically.  The  examinations 
showed  that  whether  or  not  the  diseased  material  \'^\  to  the  bees 
contained  Bacillus  <rl>-,i  or  Streptococcus  apis  these  species  were 
in  the  early  stages  of  the  disease  either  absent,  or  present  in  -mall 
numbers  only.  It  is  quite  evident  that  the  disease  was  not  pro- 
duced l>y  species  of  bacteria  which  were  absent  at  this  early  stage 
of  the  disease. 

Continuing  the  bacteriological  study  of  the  larva?  in  the  early 
stages  of  the  disease,  some  new  species  were  found  to  he  present. 
One  bacterium  especially  is  frequently  encountered.  This  species 
i-  a  small,  -lender  rod.  apparently  nonmotile  and  non-pore  bearing. 
It  i-  t"  V  known  by  the  name  Bacterium  eurydice.  It-  description 
will  appear  in  a  later  publication.  Experimental  colonic-  have  been 
fed  pure  culture-  of  this  species,  !>ut  no  disease  has  been  produced. 
Tentatively,  therefore,  this  species  is  not  to  be  regarded  as  the  cause 
of  European  foul  brood. 

Two  oiher  species  of  bacteria  mighl  he  mentioned  here  as  being 
of  interest  in  connection  with  the  study  of  the  brood  diseases.  The 
lir-t  to  be  mentioned  is  a  motile,  spore-bearing,  easily  cultivatable 
rod.  It  is  to  receive  the  name  Bacillus  orphi  "••>•.  It  also  will  he  de- 
scribed later.    This  species  is  occasionally  found  in  very  large  num.- 


4  TI-IK    CAISi:    OF    EUROPEAN    FOUL   BROOD. 

bers  in  samples  of  European  foul  brood.  Feeding  it  in  pure  cultures 
has  so  far  given  negative  results.  This  organism,  then,  can  also 
be  eliminated  tentatively  from  the  list  of  possible  causes  of  this 
disease.  The  other  species,  mentioned  by  Lambotte  in  1902  as 
Bacillus  iik -xi ntci icns  vulgaris^  may  be  said  to  belong  to  a  group  of 
bacteria  found  quite  widely  distributed  in  the  apiary.  Its  infre- 
quency  in  diseased  brood  and  its  occurrence  in  small  numbers  readily 
eliminates  this  species  from  the  list  of  possible  causes.  Most  of  the 
bacteria  that  are  met  with  in  the  study  of  European  foul  brood  were 
therefore  excluded  tentatively  from  the  list  of  possible  causes  of  the 
di-ease. 

Idie  possibility  of  an  ultramicroscopic  virus  was  also  considered. 
Brood  sick  or  dead  of  European  foul  brood  were  removed  from  the 
combs  and  crushed.  An  aqueous  suspension  of  this  diseased  material 
was  then  made  in  boiled  water  and  filtered  with  the  Berkefeld  filter. 
The  filtrate  remained  clear  when  incubated  at  different  temperatures 
and  cultures  made  from  it  produced  no  growth.  Separate  filt rations 
have  been  made  of  diseased  brood  received  from  various  localities,  but 
in  no  instance  where  healthy  colonies  were  fed  filtrate  obtained  in 
this  way  was  European  foul  brood  produced.  The  results  of  the  ex- 
periments therefore  justify  the  tentative  conclusion  that  there  is  no 
filterable  virus  in  European  foul  brood  capable  of  producing  the 
disease.  To  this  extent,  then,  has  the  possibility  of  an  ultramicro- 
scopic virus  been  eliminated. 

Having  thus  tentatively  eliminated  all  the  microscopically  visible 
organisms  except  Bacillus  Y  from  the  list  of  possible  causes  and  like- 
wise eleminated  the  probability  of  an  ultramicroscopic  virus,  the 
tentative  conclusion  was  naturally  reached  that  this  remaining  micro- 
organism probably  plays  an  important  role  in  the  etiology  of  Euro- 
pean foul  brood.  Such  a  conclusion  was  all  the  more  imperative 
since  this  organism  had  been  encountered  so  frequently  in  the  brood 
of  this  disease  and  since,  moreover,  there  had  been  no  other  factor 
observed  to  which  the  exciting  cause  could  be  attributed. 

SYMPTOMS    MANIFESTED    BY    l.ARWE    SICK    OF    EUROPEAN    FOUL   BROOD. 

This  conclusion  led  to  a  more  extended  study  of  this  microorganism 
in  the  disease  produced  experimentally.  The  presence  of  disease 
can  usually  be  detected  in  the  experimental  colony  during  the  week 
that  the  feeding  is  begun.  The  first  indication  of  it  may  be  that 
only  a  portion  of  a  larva  is  seen  in  a  cell  (fig.  1).  the  remaining  por- 
tion having  been  removed  by  the  bees.  Aside  from  an  observation 
of  this  kind  the  earliest  indication  one  get-  from  the  macroscopic 
(gioss)  examination  is  that  sick  larva1  are  found  among  the  uncapped 
brood.  One  should  acquaint  himself,  therefore,  with  certain  symp- 
toms or  signs  manifested  by  sick  larva1  during  the  course  of  the  dis- 


I  Hi  ,  \r-i  01  li  R0P1  \N  FOUL  BROOD. 


,..,.,.  bj  which  ii-  presence  can  be  diagnosed  while  the  larva?  are  stiU 
alive.    Some  of  these  will  now  be  considered. 


I      Larra    aica    of    European    fool  Fv     2      Healthj   larva  of  the  age  repre 

.1,    partlj    removed    bj    the    I  »nted  In  ngnrc  I      (Original  I 

.  <  >rii.-i n:i  1   I 

The  length  of  time  that  a  developing  bee  is  sicfc  of  European  foul 
brood  i-  variable.     It  can  be  stated  in  a  general  way  thai  the  three 


■to,  s     sick  larva  of  the  age  rapreaented 
in  Bgnrea  2  :m«i  t      (Original.) 

.lay-  ju-t  preceding  the  time  when 
a  larva  would  ordinarily  be  capped 
is  the  most  favorable  period  for 
making  a  diagnosis  from  the  gross 
examination  alone. 

When  healthy  larva'  of  the  age 
represented  in  figures  -.  3,  i.  and 
5  are  slightly  magnified  a  peristal- 


sirk    larra    with    re  if    of    call 
removed      (Original  i 


6  THE    CAUSE    OF   EUROPEAN    FOUL  BROOD. 

sislike  motion  of  theii  bodies  is  easily  -ecu.  hut  larvae  of  this  same 
age  when  sick  frequently  exhibit  a  marked  peristalsislike  motion  of 
their  bodies  which  can  be  easily  seen  with  the  unaided  eye.     Some- 


FlG.  5. — Sick  Larva  which  is  more  trans- 
parent than  a  healthy  larva  of  the 
same  age.      (Original.) 


Fh..  (j.  —  Healthy  larva' with  dorsal  wall 
turned  toward  the  observer  showing 
the  narrow  transparent  area  along  the 
median  dorsal   wall.      (Original,  i 


times  the  color  of  the  larva?  assists  in  the  selection  of  those  that  are 
diseased.  If.  instead  of  the  glistening  white  or  bluish-white  appear- 
ance of  healthy  larvae,  one  observes  some  that  are  more  transparent 

(fig.  5),  or  that  possess  a  very 
slight  yellowish  tint,  frequently 
such  larva>  are  diseased.  In  the 
absence  of  the  exaggerated  peri- 
stalsislike movement,  however, 
other  tests  should  be  applied,  as 
the  color  symptom  is  at  times  de- 
ceptive. 

Figures  6  and  7  represent  older 
larvae  than  the  preceding.  These 
have  turned  themselves  in  the  cell 
so  as  to  present  a  dorsal  portion 
to  the  observer.  The  narrow  and 
quite  transparent  area  frequently 
seen  along  the  dorsal  median  line 
of  a  larva  serves  often   a  useful 

FIG.  7.— Sick  larva   of  the  age  represented      pUrpose   ill   the  diagnosis  of  ElirO- 
in  figure  G.      (Original,  i  *        l  ° 

pean  toul  brood.  In  a  healthy 
larva  (fig.  6)  a  pollen-colored  intestinal  mass  is  frequently  plainly 
visible  through  this  transparent  dorsal  area.  Microscopically  thi> 
mass  is  easily  demonstrated  to  be  largely  pollen.     If.  however,  upon 


I  ill     CAU8E  OF  El  ROP1  w    FOUL    BH<  l  D.  7 

inspection  this  intestinal  mass  appears  white  or  yellowish  white,  the 
presence  of  European  foul  brood  is  almost  certain.  A  modification 
of  this  simple  inspection  method  may  often  be  profitably  used.  This 
consists  simply  in  turning  the  larva  in  the  cell  with  a  pair  of  forceps 
until  the  median  dorsal  line  is  exposed  t<»  the  observer. 

More  frequently  still,  it  will  be  found  advantageous  to  remove  the 
larva  From  the  ©all  with  the  forceps.  With  a  little  care  this  can  be 
done,  leaving  the  larva  intact.  It'  the  larva  is  diseased  and  the  dis- 
ease is  sufficiently  advanced,  a  uliiti~.li  intestinal  content  can  very 
often  U'  plainly  observed.  In  response  to  the  muscular  action  <»f  the 
larva  this  mass  is  frequently  seen  to  be  moved  t<>  and  fro. 

v  posrnvi  ii  -i  roa  rm   ihm  \m    i\  living  ulrvm. 

There  is  a  sign  represented  in  figure  s  which,  in  the  experience 
of  the  writer,  has  proven  thus  far  to  be  a  positive  symptom  of  Euro- 
pean foul  brood.  When  the  aire  and  condition  of  the  diseased  larva 
are  favorable  and  these  frequently  are— the  sign  can  be  quite 
easily  and  conveniently  demonstrated  in  this  way:  Select  a  larva  to 
be  tested,  approximately  of  the  age  represented  by  figures  •_'.  :'>.  and  I ; 
remove  it  from  the  cell  and  place  it  upon  glass,  preferably  with  a 
dark  background;  with  a  dissecting  needle  in  each  hand  and  with 
their  points  near  together,  pierce  with  both  needles  the  wall  of  the 
larva  near  it-  head,  avoiding  the  intestine;  separate  now  the  points 

of  the   needle-  SO  BS   to   tear  the   body   wall   CTOSSwise  and   continue  to 

separate  the  two  portions  of  the  larva.  If  the  larva  is  diseased  and 
one  i>  successful  in  applying  the  test,  it  will  be  found  that  the  in- 
testinal  content  will  be  -tripped  from  and  pulled  out  of  the  posterior 
and  blind  end  i  A.  fig.  1<M  of  the  canal,  obtaining  results  as  repre- 
sented in  figure  s.  In  case  of  living,  healthy  larva'  the  intestinal 
content  can  not  be  removed  in  this  way. 

This  mass  thus  removed  from  the  intestine,  if  examined  micro- 
scopically, will  Ih1  found,  in  general,  to  consist  of  a  white  or  slightly 
yellowish-white  mass  along  the  longitudinal  axis.    This  central  mass 

surrounded  by  a  substance  which  is  more  or  less  transparent  and 
mucuslike  in  appearance.  The  appearance  of  this  outer  portion, 
however,  will  vary  in  detail,  depending  in  a  great  measure  upon  the 
stage  of  the  disease  when  the  examination  is  made. 

The  force  which  i-  applied  in  pulling  the  ma—  from  the  intestine 
frequently  causes  this  enveloping  substance  to  -t retch  and  the  in- 
closed whitish  substance  to  break  into  segments  a-  represented  in  a 
of  figure  8.  This  is  an  earlier  stage  of  the  disease  than  that  repre- 
sented in  either  h  or  c  of  the  same  figure. 
3S1710— Cir.  167—12 2 


THE   CAUSE   OF   EUROPEAN    FOUL  BROOD. 


Pig.  S. 


-The  intestinal  content  removed  from  larvffi  sick  of  European  foul  brood  but  n 
yet  dead  of  the  disease.      (Original.) 


Till.   (   M  BE   "I     M  R0P1  vn    I  "i  L   BBOOD.  9 

It'  the  disease  ia  more  advanced  than  either  stage  represented  in 
figure  s  when  this  test  ia  applied,  a  portion  of  the  intestinal  content 

may  How  out  in  the  for f  a  sac,  the  wall  of  which  is  eery  easily 

broken.  \\" I »*•  i »  broken  the  content  of  this  saclike  structure  will  flo* 
out  as  a  rather  thin  whitish  or  yellow  ish  w  hite  fluid  containing  small 
whitish  granules  thai  vary  in  size.  If  the  disease  is  far  advanced 
and  tlic  larva  probably  dead,  the  enveloping  substance  of  the  ii 
tinal  contenl  is  so  easily  broken  that  often  only  whitish  or  yellow  ish- 
white  fluid  with  its  granular  content  flows  from  the  ruptured  wall  of 
the  Ian  a. 

Figures  2  and  6  represent  healthy  larva',  and  at  these  ages  the  seg- 
ments of  the  body  are  strongly  marked  off.     Living  larva'  at  thi 
ages,  if  suffering  from  European  foul  brood,  frequently  -how  these 
markings  less  distinctly  as  represented  in  figures  3,  I.  and  7.    This 
sign, too, may  asssist  in  the  -elec- 
tion of  larva-  that   are  suspected 

of  being  diseased. 

TIIK  V  VI  I  E  OF  EARLY  SYMPTOMS   IN  THI 
HI  L6R08I8  <>i    I  I  BOPBAfl  FOUL  BBOOD. 

These  symptoms  of  European 
foul  brood  are  some  of  the  more 
important  ones  that  are  observed 
in  sick  larva'  or  in  those  only 
recently  dead.  They  are  espe- 
cially valuable  in  the  study  of 
the  disease  in  the  experimental     r     e     bmoim  piuton  ,in  a  stained  a 

colony.    They  have  not  been  used         preparation  rroni  sick  larva;  al  stage  rep- 
.  •    .  resented  In  a  -      (Original.) 

by    the   apiarist    tor   making   a 

diagnosis.  The  symptoms  of  European  foul  brood  that  have  been 
looked  for  by  the  bee  keeper  for  the  most  part  are  the  evidences  of 
disease  which  obtain  a-  a  result  of  the  death  of  the  brood.  The 
post-mortem  symptoms  a-  manifested  by  the  dead  larvae  themselves 
have  been  the  mosl  positive  evidences  used  by  the  bee  keeper  in 
diagnosing  the  disease.  I;  is  hoped,  however,  that  when  they  are 
well  learned,  the  symptoms  of  European  foul  brood  observed  in 
living  larva'  and  in  those  very  recently  dead  may  prove  of  value  in 
the  apiary  as  well  as  in  the  experimental  colony. 

Practically  all  the  later  symptoms  of  European  foul  brood  have 
also  been  observed  during  the  course  of  the  disease  in  the  experi- 
mental colony.  This  fact  i-  used  as  evidence  that  the  disease  which 
was  produced  in  the  experimental  colony  was  the  same  a-  that  encoun- 
tered in  the  apiary.  Since  the  diseased  material  for  making  the 
inoculation-  ha-  been  received  from  various  source-  and  the  disease 
produced  was  apparently  the  same  in  every  case,  the  conclusion  that 


10  THE   CAUSE   OF  EUROPEAN   FOUL  BROOD. 

there  is:  but  one  disease  present  in  the  condition  which  is  being  called 
European  foul  brood  is,  therefore,  still  further  confirmed. 

MICROSCOPIC    STUDIES    OF    DISEASED   LARY.E. 

Returning  now  to  the  discussion  of  European  foul  brood  in  the 
earlier  stages,  it  should  be  emphasized  that  by  a  macroscopic  exami- 
nation alone  it  is  not  always  possible  to  make  a  positive  diagnosis 
of  the  presence  or  absence  of  disease  in  a  larva.  During  the  very 
earliest  period  of  infection  it  is  impossible  from  the  gross  examina- 
tion alone  to  make  a  positive  diagnosis  of  the  presence  of  disease. 
Such  is  to  be  expected.  About  the  time  the  larva  dies  there  is  a 
period  at  which  one  can  not  always  be  sure  that  the  disease  is  present 
from  a  macroscopic  examination  alone.  Between  these  stages  there 
is  a  period  in  the  course  of  the  disease  in  the  larva?  during  which  it 
is  usually  possible  to  make  a  diagnosis  positive  from  the  gross  exami- 
nation. Since  a  macroscopic  examination  alone  is  not  always  suffi- 
cient for  making  a  positive  diagnosis,  one  looks  naturally  to  a  micro- 
scopic examination  for  assistance. 

During  the  course  of  the  disease  in  the  experimental  colony  the 
microscopic  picture  presented  in  the  examination  of  diseased  larvae 
changes  markedly.  To  begin  the  microscopic  study,  it  is  well  to 
obtain  the  intestinal  content  as  represented  in  a.  figure  8.  If  a  thin 
smear  is  made  of  the  white  growth-mass  of  this  content  and  stained, 
it  is  found  to  consist  almost  entirely  of  forms  represented  in  fig- 
ure 9. 

This  organism  is  the  one  that  the  writer  referred  to  in  an  earlier 
paper  as  "  Bacillus  F."  All  attempts  to  cultivate  this  new  species 
on  artificial  media  have  thus  far  l>een  unsuccessful.  Since  consider- 
able information  has  now  been  obtained  concerning  this  organism 
the  specific  name  "  pin  ton  "  is  now  substituted  for  the  "  F  "  in  the 
term  "Bacillus  F"  and  the  species  will  now  be  known  as  Bacillus 
pluton.  This  organism  is  an  unusual  one  and  the  classification  has 
not  yet  been  definitely  determined.  The  generic  term  "  Bacillus,"' 
therefore,  may.  and  probably  will,  be  changed  later. 

At  the  stage  of  the  disease  represented  in  a,  figure  8,  the  majority 
of  the  individuals  of  this  new  species  in  general  appear  in  stained 
preparations  to  be  pointed  at  the  ends  (fig.  9).  Some  show  both 
ends  rather  sharply  pointed,  others  show  only  one  end  so  pointed, 
the  other  end  being  rounded,  while  still  others  show  both  ends 
rounded.  The  individuals  having  this  general  form  vary  much  in 
size.  They  are  as  a  rule  1  \i  or  less  in  length,  the  breadth  being 
about  one-half  the  length.  Forms  in  pairs  frequently  occur  in  a 
smear  preparation  made  at  this  stage  of  the  disease.  These  paired 
forms  vary  markedly  in  size  and  shape.     (See  fig.  9.)     Accompany- 


I  II  I     CAUS1     "I     M   ROlM   \N     I  «>i   i.    UR(K  ID. 


11 


bug  />>"  illus  phit.    .  /:    terium  enri  P    frequently  found  at  this 

stage  of  the  disease,  but  in  comparatively  small  numbers. 

If  the  intestinal  content  in  a  later  stage  of  the  disease  for  exam- 
ple, that  represented  in  6,  lii,r|ip'  B  is  examined  microscopically, 
Bacillus  pin  ton  is  still  found  in  verj  targe  numbers,  and  Bacterium 
eurydicx  when  present  will  be  relatively  veri  much  increased  in  num- 
bers. V  similar  examination  of  the  intestinal  content  represented  in 
r.  figure  s.  will  usually  show  Bacillus  pint  on  in  large  numbers,  Bac- 
terium eurydin  in  increased  numbers,  and  in  addition  one  may  find 
Hug  alri'i  in  comparatively  small  numbers. 


I  10  in.it i.-  drawing  representing  o   long  n  •  <(  n   larra  at  nn  enrlr 

■  >f  infection.     The  position  of  the  li  »m,  BaviUut  pluton,  la  aloi 

near  the  perltropblc  membrane.     (Original 

By  examining  the  fluid  mass  which  flows  from  the  body  of  a 
Larva  when  the  disease  is  far  advanced  and  Wie  body  wall  is  broken, 
pne  usually  find-,  together  with  Bacillus  pluton,  bacteria  of  different 
species  in  considerable  numbers. 

From  this  point  on  in  the  decay  of  the  larva'  the  relative  propor- 
tion of  the  different  microorganisms  present  varies  markedly.  When 
Bacillus  nl'-ii  is  present  it  increases  verj  rapidly  in  proportion  to 
the  other-.  This  rapid  increase  of  Bacillus  alvei  in  the  larvae  after 
the  death  of  the  larva;  account-  in  a  large  measure  for  the  frequency 
with  which  this  species  is  mentioned  in  report.-  on  this  disease. 


12  THE   CAUSE   OF   EUBOPEAN   FOUL  BROOD. 

Id  living  larv;v.  therefore,  in  which  European  foul  brood  can  be 
diagnosed  from  gross  examination,  it  is  found  that  bacteria  usually 
accompany  BacUlus  phiton.  This  fact  made  desirable  the  study  of 
the  diseased  larvae  in  still  earlier  stages  of  the  infection,  i.  e.,  during 

the  period  of  incubation.  This  was  done  culturally  in  part,  but  prin- 
cipally by  fixing  and  sectioning  the  younger  larvae  from  strongly 
infected  experimental  colonies.  From  such  sections  it  was  observed 
that  BacUlus  pluton  was  the  first  invader  of  the  healthy  larvae. 

Figure  10  represents  schematically  the  condition  in  the  larva'  at 
an  early  stage  of  infection.  Tn  this  figure  fg  represents  the  foregut; 
iikj  the  midgut,  and  Kg  the  hindgut.  At  this  age  of  the  larvae  the 
posterior  end  of  the  midgut  is  closed,  as  represented  at  b.  Tn  the 
same  figure,  m  represents  that  portion  of  the  intestinal  content  lying 
in  contact  with  the  wall  of  the  intestine:  /.  the  central  portion  of  the 
drawing,  represents  the  food  taken  at  this  age;  and  p  represents 
what  seems  to  be  a  peritrophic  membrane  between  the  enveloping 
substance,  m,  and  the  paplike  food  substance.  /.  of  the  midgut. 

In  the  growth  of  Bacillus  phiton  this  parasite  very  early  takes  a 
position  along  the.  peritrophic  membrane  p.  and  just  central  to  it 
(fig.  10).  At  this  early  stage  of  its  growth  this  microorganism  pre- 
sents in  general  an  appearance  of  being  rod  shaped  with  a  strong 
tendency  to  <rrow  in  chains.  As  the  disease  advances  and  the  growth- 
mass  of  this  organism  increases,  the  central  portion  of  the  lumen  of 
the  intestine  becomes  filled  by  a  solid  growth  which  is  made  up  very 
largely  of  Bacillus  pluton.  During  this  stage  of  the  disease  the  con- 
tent can  be  removed  from  the  posterior  blind  end  of  the  midgut,  as 
shown  in  figure  8.  The  relation  of  the  central  growth-mass  to  the 
surrounding  mucuslike-appearing  mass  represented  in  a,  b,  and  c 
of  figure  8  is  well  demonstrated  microscopically  by  sectioning  these 
intestinal  masses. 

From  the  studies  made  thus  far  it  would  seem  that  Bacillus  phiton 
is  easily  killed  by  heat. 

PROBABLE    EXPLANATIONS    OF    ERRORS    AS    TO    THE     EXCITING     CAUSE    OF 

EUROPEAN  FOUL  BROOD. 

It  is  quite  probable  that  others  at  different  times  have  observed  this 
new  species.  I!<n  illns  pluton,  but  have  failed  to  differentiate  it  from 
bacteria  which  were  present  and  which  appeared  in  the  cultures 
made,  leading  them  thus  to  erroneous  statements  concerning  the  dis- 
ease and  its  exciting  cause.  For  example.  William  R.  Howard  may 
have  seen  this  organism  microscopically  in  his  so-called  "  black 
brood."  but  failed  to  differentiate  it  from  some  bacterium — BacUlus 
milii  or  Bacillus  alvei — which  he  cultivated  on  artificial  media. 
Burri  may  have  seen  it  in  the  so-called  "sour  brood  "  and  mistaken  it 


1  III     CAU81    01     m  BOP]  w    FOUL   BBOOD.  1  .''> 

for  the  u  ffuntheri- forms*1  which  he  observed  in  hia  cultures.  Maa  Ben 
mentions  some  difficulty  experienced  at  times  in  obtaining  Strep 
coccus  apis  from  brood  which  on  microscopic  examination  seemed  to 
contain  this  bacterium.  To  explain  this  difficulty,  he  advanced  the 
supposition  that  the  Streptococcus  was  probabh  killed  by  acid  pro- 
duced by  itself.  The  difficulty  probably  could  be  as  "''II  explained 
by  supposing  thai  Maassen  failed  to  differentiate  this  parasite  from 
the  bacterium  which  he  cultivated  and  described  as  Streptocod  us  apis. 

is     Mil  i:i      Mci;i      iiiw     ONI      DISJ   \-i      in      nil      CONDITION     KNOWN      \- 
l  l  Ri  >PE  w    FOUL  BBOOD  i 

The  question  now  arises  whether  or  nol  there  is  more  than  one 
disease  in  the  condition  now  known  a-  European  foul  brood.  In 
Switzerland  and  in  Germany  there  has  been  a  tendency  to  diagnose 
the  diseased  brood  in  which  Bacillus  alvei  is  found  as  the  foul  brood 
of  Cheshire  and  Cheyne  and  the  diseased  brood  in  which  Strepto- 
apis  is  found  as  "sour  brood."  From  the  facts  at  hand  the 
writer  is  strongly  inclined  to  believe  that  these  two  conditions  are 
only  the  one  disease,  known  in  America  a-  European  foul  brood. 
Enough  evidence  has  not  yet  been  obtained,  however,  to  -peak  with 
complete  positiveness  on  this  point. 

\-  secondary  invaders  some  of  the  specie-  of  bacteria  mentioned 
in  this  paper  may  and  probably  do  exert  an  influence  on  the  course 
of  the  disease  in  the  larva  and  in  the  colony.  To  what  extent  these 
bacteria  modify  the  disease  is  yet  to  lie  determined.  Should  it  he 
found  that  Bacillus  alvei  actually  causes  an  infection-  brood  disease, 
then  such  a  disease  should  1h>  called  European  foul  brood,  and  the 
disease  caused  by  Bacillus  pluton  would  have  to  Ik-  differentiated 
from  it. 

Further  detail-  will  not  he  given  in  thi>  preliminary  announce- 
ment hut    will  he  included   in   more  technical   paper-  which  are  being 

prepared. 

-t  M  m  un     \M>  CON<  ii  BI0N8. 

The  -tep-  taken  in  the  writer'-  endeavor  to  find  the  cause  of  Kuro- 
pean  foul  brood  may  l>c  briefly  summarized  a-  follow-: 

lit  Hit,  it  his  alvei)  which  has  been  so  generally  spoken  of  as  the 
cause  of  foul  brood,  was  isolated  from  diseased  brood,  and  pure  cul- 
ture- of  the  organism  in  both  the  vegetative  and  -pore  forms  were 
repeatedly  \\-A  to  colonies  of  healthy  U-e-  with  the  result  that  foul 
brood  was  not  produced  in  any  instance.  This  fact  cast  a  suspicion 
that  Bacillus  alvei  was  probably  not  the  cause  of  a  disease. 

(2)  By  a  study  of  many  larva-  in  -ample-  of  European  foul  brood 
it  was  frequently  found  that  there  were  larvae  apparently  dead  of  the 
disease  that  contained  Bacillus  alvei  only  in  small  numbers  or  not  at 


14  THE   CAUSE   OF   EUROPEAN    FOUL   BROOD. 

all.     This   increased   the   suspicion   that    Bacillus  <>/>■<;   was  not   the 
exciting  cause  of  the  disorder. 

(3)  In  1907  the  writer  proved  that  by  feeding  pure  cultures  of 
Bacillus  larval  to  healthy  bees  American  foul  brood  could  be  produced. 
This  fact  still  further  emphasized  the  doubt  that  was  already  enter- 
tained concerning  the  possibilities  of  Bacillus  alvei  in  the  etiology  of 
European  foul  brood. 

(4)  By  feeding  diseased  larva'  to  healthy  colonies  it  was  found  that 
European  foul  brood  could  be  artificially  produced,  showing  that  this 
disease,  too,  could  be  produced  by  feeding,  and  that  the  virus  was 
contained  in  the  diseased  brood. 

( ."> )  The  sick  larvae  of  the  disease  thin  artificially  produced  were 
frequently  found,  when  examined,  to  be  free  from  Bacillus  at 
This  evidence,  too,  was  damaging  to  the  theory  that  Bacillus  alvei  is 
the  cause  of  a  brood  disease. 

(6)  Bacillus  aire/  in  this  way  was  tentatively  eliminated  from  the 
list  of  possible  exciting  causes  of  European  foul  brood.  In  a  quite 
similar  manner  the  other  bacteria — Streptococcus  apis.  Bacillus  me- 
sentericus  vulgaris,  Bacillus  orpkeus,  and  Bacteiiuni  earn/dice — were 
likewise  eliminated  from  the  list. 

(7)  Considerable  quantities  of  filtrate  from  aqueous  suspensions  of 
crushed  diseased  larvae  were  fed  to  healthy  colonies  and  in  no  instance 
was  European  foul  brood  produced.  This  eliminated  tentatively  the 
probability  of  there  being  an  ultramicroscopic  virus  in  European  foul 
brood  capable  of  producing  the  disease. 

(8)  Bacillus  pluton,  therefore,  was  the  only  factor  that  was  not 
so  eliminated  from  the  list  of  possible  exciting  causes  of  the  disease 
and  became  thus  the  probable  exciting  cause  of  European  foul  brood 

(9)  "When  this  organism  was  studied  in  larva1  in  which  the  disease 
could  be  suspected  by  inspection  alone,  one  or  more  species  of  bacteria 
were  sometimes  found  to  be  present  als"o.  These,  when  present,  how- 
ever, occurred  in  relatively  small  number-. 

(10)  The  disease  was  then  studied  in  a  still  earlier  stage;  i.  e.. 
before  its  presence  could  be  detected  by  gross  examination  of  the 
larva1.  This  was  done  by  cultures  in  part,  but  principally  by  fixing 
and  sectioning  larva  during  the  incubation  period  of  the  disease. 
This  study  demonstrated  that  in  the  production  of  the  disease  Bacillus 
pluton  was  the  first  invader  of  the  healthy  larvae. 

It  will  be  noticed,  therefore,  that  in  the  determination  of  the  pri- 
mary exciting  cause  of  European  foul  brood  two  objects  were  accom- 
plished:  (1)  All  the  factors  in  the  list  of  possible  exciting  causes  of 
the  disease  were  eliminated  except  the  one  organism  Bacillus  pluton, 
and  (2)  by  the  study  of  infected  larva1  soon  after  the  infection  took 
place,  this  parasite  was  found  to  be  the  first  invader. 


I  II  v     CAUSI     i»i     1  l'KOl'1   W     FOUL    BROOD. 


i:» 


As  b  conclusion,  it  i-  the  belief  of  the  writer  thai  sufficient  evidence 
has  noM  been  obtained  to  justify  the  statement  thai  Bacillus  pluton 
is  the  primary  exciting  cause  oi  a  brood  disease.    Thia  brood  dise 
is  now  generall)  known  in  America  as  European  foul  brood.     Hi; 
opinion  is  rendered  in  accordance  with  view-  no*  generally  accepted 
relative  to  the  etiology  of  animal  disea 

There  are,  then,  three  principal  brood  di  •  Two  of  thi 

American  foul  brood,  caused  l»\  Bacillus  larva1,  and  European  foul 
brood,  caused  by  Bacillus  pluton  are  known  l<>  be  infectious.  Prom 
these  two  diseases  there  must  be  differentiated  the  third  one,  an 
apparently  noninfectious  disorder,  the  so-called  w  pickled  brood." 
Larva?  dead  of  this  latter  disease  are  practically  free  from  micro 
organisms.     The  exciting  cause  of  thia  disorder  is  not  yet  known. 

Approved : 

.1  \mi  a  Wilson, 

s  ■ ,  ij  of  .  I  '/'  U  ult  in  . , 

Washington,  D.  ('..  March   28,  1912. 


\M>I  i  [ON  \1.  COPIES  ol 
I  procured  bom  I 

•  i    Printing 
w  i  bJngt 


UNIVERSITY  OF  FLORIDA 


" 


3  1262  09216  5793 


